Inhibitors of the interaction between HMGB polypeptides and toll-like receptor 2 as anti-inflammatory agents

ABSTRACT

The invention features a method of treating an inflammatory condition in an individual, comprising administering an agent inhibits the interaction between a Toll-like receptor 2 (TLR2) and a high mobility group B (HMGB) polypeptide to the individual. The invention also features methods for identifying agents that inhibit the interaction between TLR2 and HMGB.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/456,947, filed Jun. 6, 2003. The entire teachings of the aboveapplication are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant GM 62508KTfrom the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Inflammation is often induced by proinflammatory cytokines, such astumor necrosis factor (TNF), interleukin (IL)-b 1α, IL-1β, IL-6,platelet-activating factor (PAF), macrophage migration inhibitory factor(MIF), and other compounds. These proinflammatory cytokines are producedby several different cell types, including immune cells (for example,monocytes, macrophages and neutrophils), and non-immune cells such asfibroblasts, osteoblasts, smooth muscle cells, epithelial cells, andneurons. These proinflammatory cytokines are part of an inflammatorycytokine cascade that contributes to the occurrence of inflammation. Inaddition to mediation of inflammation, early proinflammatory cytokines(e.g., TNF, IL-1, etc.) induce the late release of high mobility groupbox 1 (HMGB1; also known as HMG-1 and HMG1), a protein that accumulatesin serum and mediates delayed lethality and further induction of earlyproinflammatory cytokines.

HMGB1 was first identified as the founding member of a family ofDNA-binding proteins termed high mobility group box (HMGB) proteins thatare critical for DNA structure and stability. It was identified nearly40 years ago as a ubiquitously expressed nuclear protein that bindsdouble-stranded DNA without sequence specificity. The HMGB1 protein hasthree domains: two DNA binding motifs termed HMGB A and HMGB B boxes,and an acidic carboxyl terminus. The two HMGB boxes are highly conserved80 amino acid, L-shaped domains.

Recent evidence has implicated HMGB1 as a mediator of a number ofinflammatory conditions (U.S. Pat. No. 6,303,321). The delayed kineticsof HMGB1 appearance during endotoxemia makes it a potentially goodtherapeutic target, but little is known about the molecular basis ofHMGB1 signaling and toxicity.

SUMMARY OF THE INVENTION

It has been discovered that HMGB polypeptides bind Toll-like receptor 2(TLR2) and can mediate biological effects through TLR2. It has also beendiscovered that inhibition of the interaction between HMGB and TLR2 candecrease or prevent inflammation. Therefore, agents that inhibit theinteraction of an HMGB polypeptide with TLR2 can be used to treatinflammatory conditions.

Accordingly, in a first aspect, the invention features a method oftreating an inflammatory condition in an individual, comprisingadministering to the individual an effective amount of an agent thatinhibits the interaction between an HMGB polypeptide or a functionalequivalent thereof and a TLR2, with the proviso that the agent is not anantibody that binds to an HMGB1 polypeptide. In one embodiment, theagent binds a TLR2 and inhibits binding by an HMGB polypeptide orfunctional equivalent thereof.

In another aspect, the invention features a method of inhibiting therelease of a proinflammatory cytokine from a cell, comprisingadministering to the cell an effective amount of an agent that inhibitsthe interaction between an HMGB polypeptide or functional equivalentthereof and a TLR2, with the proviso that the agent is not an antibodythat binds to an HMGB1 polypeptide. In one embodiment, the agent binds aTLR2 and inhibits binding by an HMGB polypeptide or functionalequivalent thereof.

In another aspect, the invention features a method of determiningwhether an agent inhibits inflammation, comprising contacting a TLR2with the agent and an HMGB polypeptide or a functional equivalentthereof; and detecting binding of an HMGB polypeptide to the TLR2,wherein an agent that decreases binding of the HMGB polypeptide orfunctional equivalent to the TLR2 relative to a suitable control is anagent that inhibits inflammation.

In another aspect, the invention features an ex vivo method ofdetermining whether an agent inhibits inflammation, comprisingcontacting a cell comprising a TLR2 with the agent and an HMGBpolypeptide or functional equivalent thereof; and measuring release of aproinflammatory cytokine from the cell, wherein an agent that decreasesrelease of the proinflammatory cytokine from the cell relative to asuitable control is an agent that inhibits inflammation.

The present invention provides the advantage of identifying new agentsand/or methods for treating (or preventing) inflammatory conditions, aswell as methods for identifying such agents. These agents act at thelevel of the interaction between HMGB polypeptides and TLR2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a histogram of the effect of HMG1 or HMG1 B box polypeptidestimulation on activation of the NF-κB-dependent ELAM promoter (measuredby luciferase activity) in RAW 264.7 cells co-transfected with a murineMyD 88-dominant negative (+MyD 88 DN) mutant (corresponding to aminoacids 146-296), or empty vector (−MyD 88 DN). Data are expressed as theratio (fold-activation) of average luciferase values from unstimulatedand stimulated cells (subtracted for background)+SD.

FIG. 1B is a histogram of the effect of stimulation of CHO reporter celllines that constitutively express human TLR2 (open bars) or TLR4 (shadedbars) with IL-1, HMG1, or HMG1 B box on CD25 expression. Data areexpressed as the ratio (fold-activation) of the percent of CD25⁺ cellsin unstimulated and stimulated cell populations that were gated toexclude the lowest 5% of cells based on mean FL1 fluorescence.

FIG. 1C is a histogram of the effect of administration of anti-RAGEantibody, anti-TLR2 antibody, anti-RAGE antibody and anti-TLR2 antibodytogether, or IgG on HMG1-mediated TNF release (measured as a percent ofTNF release in the absence of antibody) in RAW 264.7 cells.

FIG. 2A is a histogram showing the effects of administration of HMGB1(expressed in E. coli) to human embryonic kidney cells (HEK cells)stably transfected with pcDNA (HEK-PCDNA), TLR2 (HEK-TLR2), or TLR4(HEK-TLR4). HEK cells over-expressing TLR2, TLR4, or vector alone (PCDNA) were plated in a 24-well culture plate and treated with HMGB1(expressed in E. coli) at the concentrations indicated for 18 hours.IL-8 release in conditioned supernatants was measured by ELISA. Datashown are mean±SEM, n=3 or more.

FIG. 2B is a histogram showing the effects of administration of HMGB1(expressed in Chinese hamster ovary (CHO) cells) to human embryonickidney cells (HEK cells) stably transfected with TLR2 (HEK-TLR2). HEKcells over-expressing TLR2 were plated in a 24-well culture plate andtreated with HMGB1 (expressed in CHO cells) at the concentrationsindicated for 18 hours. IL-8 release in conditioned supernatants wasmeasured by ELISA. Data shown are mean±SEM, n=3 or more.

FIG. 3A is a histogram showing the specificity of HMGB1-induced IL-8release in HEK-TLR2 cells under various treatment conditions, assessedby measuring IL-8 release from the cells. HEK-TLR2-expressing cells in a24-well plate were stimulated with HMGB1 (trypsinized or intact, in theindicated amounts) or LPS, as indicated for 18 hours, with or withoutIgG-purified anti-HMGB1 antibody or non-immune IgG added, as indicated.LPS was sonicated for 20 minutes before use. N=3 separate experiments.

FIG. 3B is a histogram showing the specificity of HMGB1-induced IL-8release in HEK-TLR4 cells under various treatment conditions, assessedby measuring IL-8 release from the cells. HEK-TLR4-expressing cells in a24-well plate were stimulated with HMGB1 (intact, in the indicatedamounts) or LPS as indicated for 18 hours (sonicated for 20 minutesbefore use). N=3 separate experiments.

FIG. 4A is a histogram showing the effect of dominant negative MyD 88 onNF-κB-dependent luciferase activity. Sub-confluent RAW 264.7 cells(over-expressing either wild type or mutant MyD88 and an NF-κB-dependentluciferase reporter) in 24-well plates were stimulated with HMGB1 (100ng/ml) for 5 hours. Cells were then harvested and luciferase activitywas measured. All transfections were performed in triplicate, repeatedat least three times, and a single representative experiment is shown.Data are expressed as the ratio (fold-activation) of average luciferasevalues from un-stimulated and stimulated cells (subtracted forbackground)±SD.

FIG. 4B is a histogram showing the effect of HMGB1 or IL-1 onNF-κB-dependent CD25 expression in CHO cells transfected with eitherTLR2 or TLR4. CHO/TLR2 and CHO/TLR4 cells were seeded into 24-welltissue culture plates and used at 90% confluence. Cells were stimulatedwith IL-1 (10 ng/ml) or purified HMGB1 (100 ng/ml) for 18 hours.Following stimulation, cells were stained with a PE-labeled anti-CD25monoclonal antibody and surface expression of CD25 was measured by flowcytometry. Data are expressed as the ratio (fold-activation) of thepercent of CD25 positive cells in un-stimulated and stimulated cellpopulations that were gated to exclude the lowest 5% of cells based onmean FL1 fluorescence.

FIG. 5A is a scanned confocal microscopic image of RAW 264.7 cellsadministered FITC-labeled HMGB1. Macrophage-like RAW 264.7 cells wereplated in an 8-well slide chamber and used at 70% confluence. Cells wereincubated with FITC-labeled HMGB1 at 1 μg/ml for 15 minutes at 37° C.Cells were then washed 3 times with PBS and fixed for 15 minutes at roomtemperature in 4% paraformaldehyde-PBS solution (pH 7.2). After fixing,cells were washed with PBS once and mounted for viewing by fluorescentconfocal microscopy. Magnification: ×40.

FIG. 5B is a scanned confocal microscopic image of RAW 264.7 cellsadministered anti-TLR2 antibody and FITC-labeled HMGB1. Macrophage-likeRAW 264.7 cells were plated in an 8-well slide chamber and used at 70%confluence. Cells were pre-incubated with anti-TLR2 antibody at 1 μg/mlfor 20 minutes at 37° C. in Opti-MEM I medium, and then FITC-labeledHMGB1 was added at 1 μg/ml and the cells were incubated for anadditional 15 minutes at 37° C. Cells were washed 3 times with PBS andfixed for 15 minutes at room temperature in 4% paraformaldehyde-PBSsolution (pH 7.2). After fixing, cells were washed with PBS once andmounted for viewing by fluorescent confocal microscopy. Magnification:×40.

FIG. 5C is a scanned confocal microscopic image of RAW 264.7 cellsadministered anti-TLR4 antibody and FITC-labeled HMGB1. Macrophage-likeRAW 264.7 cells were plated in an 8-well slide chamber and used at 70%confluence. Cells were pre-incubated with anti-TLR4 antibody at 1 μg/mlfor 20 minutes at 37° C. in Opti-MEM I medium, and then FITC-labeledHMGB1 was added at 1 μg/ml and the cells were incubated for anadditional 15 minutes at 37° C. Cells were washed 3 times with PBS andfixed for 15 minutes at room temperature in 4% paraformaldehyde-PBSsolution (pH 7.2). After fixing, cells were washed with PBS once andmounted for viewing by fluorescent confocal microscopy. Magnification:×40.

FIG. 6A is the amino acid sequence of a human HMGB1 polypeptide (SEQ IDNO:1).

FIG. 6B is the amino acid sequence of rat and mouse HMGB1 (SEQ ID NO:2).

FIG. 6C is the amino acid sequence of human HMGB2 (SEQ ID NO:3).

FIG. 6D is the amino acid sequence of a human, mouse, and rat HMGB1 Abox polypeptide (SEQ ID NO:4).

FIG. 6E is the amino acid sequence of a human, mouse, and rat HMGB1 Bbox polypeptide (SEQ ID NO:5).

FIG. 7A is the nucleic acid sequence of HMG1L10 (SEQ ID NO:9) encodingan HMGB polypeptide.

FIG. 7B is the polypeptide sequence of HMG1L10 (SEQ ID NO: 10) encodingan HMGB polypeptide.

FIG. 7C is the nucleic acid sequence of HMG1L1 (SEQ ID NO: 11) encodingan HMGB polypeptide.

FIG. 7D is the polypeptide sequence of HMG1L1 (SEQ ID NO: 12) encodingan HMGB polypeptide.

FIG. 7E is the nucleic acid sequence of HMG1L4 (SEQ ID NO: 13) encodingan HMGB polypeptide.

FIG. 7F is the polypeptide sequence of HMG1L4 (SEQ ID NO: 14) encodingan HMGB polypeptide.

FIG. 7G is the nucleic acid sequence of the HMG polypeptide sequence ofthe BAC clone RP11-395A23 (SEQ ID NO: 15).

FIG. 7H is the polypeptide sequence of the HMG polypeptide sequence ofthe BAC clone RP11-395A23 (SEQ ID NO: 16 encoding an HMGB polypeptide.

FIG. 7I is the nucleic acid sequence of HMG1L9 (SEQ ID NO: 17) encodingan HMGB polypeptide.

FIG. 7J is the polypeptide sequence of HMG1L9 (SEQ ID NO: 18) encodingan HMGB polypeptide.

FIG. 7K is the nucleic acid sequence of LOC122441 (SEQ ID NO: 19)encoding an HMGB polypeptide.

FIG. 7L is the polypeptide sequence of LOC122441 (SEQ ID NO: 20)encoding an HMGB polypeptide.

FIG. 7M is the nucleic acid sequence of LOC139603 (SEQ ID NO: 21)encoding an HMGB polypeptide.

FIG. 7N is the polypeptide sequence of LOC139603 (SEQ ID NO: 22)encoding an HMGB polypeptide.

FIG. 7O is the nucleic acid sequence of HMG1L8 (SEQ ID NO: 23) encodingan HMGB polypeptide.

FIG. 7P is the polypeptide sequence of HMG1L8 (SEQ ID NO: 24) encodingan HMGB polypeptide.

FIG. 8 is the polypeptide sequence of human TLR2 (SEQ ID NO: 46).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that HMGB biologicalactivity is mediated through binding of HMGB to Toll-like receptor 2(TLR2). Therefore, the biological activity of HMGB can be inhibited byinhibiting binding of HMGB to TLR2. This inhibition can be achieved byproviding an agent that binds TLR2 and prevents binding by an HMGBpolypeptide. Alternatively, the agent can bind the HMGB polypeptide andprevent the polypeptide from binding to TLR2. As HMGB mediates releaseof proinflammatory cytokines from a cell, agents that inhibit theinteraction between HMGB and TLR2 can be used to prevent release of oneor more proinflammatory cytokines from the cell. These proinflammatorycytokines contribute to many inflammatory conditions; therefore, agentsthat inhibit the interaction between HMGB and TLR2 can be used to treator inhibit inflammatory conditions mediated through the inflammatorycytokine cascade. The present invention also features methods ofidentifying agents that inhibit the interaction between HMGB and TLR2.Agents identified through such screening methods can be used to treat aninflammatory condition, or to inhibit release of a proinflammatorycytokine from a cell.

HMGB Polypeptides

As used herein, an “HMGB polypeptide” is polypeptide that has at least60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, and mostpreferably at least 95% sequence identity to a sequence selected fromSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:6(MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEEEDEEDEEDEEEDDDDE) (as determined, for example, using theBLAST program and parameters described herein) and increasesinflammation and/or increases release of a proinflammatory cytokine froma cell. In one embodiment, the HMGB polypeptide has one of the abovebiological activities. Typically, the HMGB polypeptide has both of theabove biological activities. The term “polypeptide” refers to a polymerof amino acids, and not to a specific length; thus, peptides,oligopeptides and proteins are included within the definition of apolypeptide. Preferably, the HMGB polypeptide is a mammalian HMGBpolypeptide, for example, a human HMGB1 polypeptide. Examples of an HMGBpolypeptide include a polypeptide comprising or consisting of thesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:6.

Other examples of HMGB polypeptides are described in GenBank AccessionNumbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429,NP_(—)002119, CAA3 1110, S02826, U00431, X67668, NP_(—)005333,NM_(—)016957, and J04179, the entire teachings of which are incorporatedherein by reference. Additional examples of HMGB polypeptides include,but are not limited to mammalian HMG1 ((HMGB1) as described, forexample, in GenBank Accession Number U51677), HMG2 ((HMGB2) asdescribed, for example, in GenBank Accession Number M83665), HMG-2A((HMGB3, HMG-4) as described, for example, in GenBank Accession NumbersNM_(—)005342 and NP_(—)005333), HMG14 (as described, for example, inGenBank Accession Number P05114), HMG17 (as described, for example, inGenBank Accession Number X13546), HMGI (as described, for example, inGenBank Accession Number L17131), and HMGY (as described, for example,in GenBank Accession Number M23618); nonmammalian HMG T1 (as described,for example, in GenBank Accession Number X02666) and HMG T2 (asdescribed, for example, in GenBank Accession Number L32859) (rainbowtrout); HMG-X (as described, for example, in GenBank Accession NumberD30765) (Xenopus), HMG D (as described, for example, in GenBankAccession Number X71138) and HMG Z (as described, for example, inGenBank Accession Number X71139) (Drosophila); NHP10 protein (HMGprotein homolog NHP 1) (as described, for example, in GenBank AccessionNumber Z48008) (yeast); non-histone chromosomal protein (as described,for example, in GenBank Accession Number O00479) (yeast); HMG 1/2 likeprotein (as described, for example, in GenBank Accession Number Z11540)(wheat, maize, soybean); upstream binding factor (UBF-1) (as described,for example, in GenBank Accession Number X53390); PMS1 protein homolog 1(as described, for example, in GenBank Accession Number U13695);single-strand recognition protein (SSRP, structure-specific recognitionprotein) (as described, for example, in GenBank Accession NumberM86737); the HMG homolog TDP-1 (as described, for example, in GenBankAccession Number M74017); mammalian sex-determining region Y protein(SRY, testis-determining factor) (as described, for example, in GenBankAccession Number X53772); fungal proteins: mat-1 (as described, forexample, in GenBank Accession Number AB00945 1), ste 11 (as described,for example, in GenBank Accession Number x53431) and Mc 1; SOX 14 (asdescribed, for example, in GenBank Accession Number AF107043) (as wellas SOX 1 (as described, for example, in GenBank Accession NumberY13436), SOX 2 (as described, for example, in GenBank Accession NumberZ31560), SOX 3 (as described, for example, in GenBank Accession NumberX71135), SOX 6 (as described, for example, in GenBank Accession NumberAF309034), SOX 8 (as described, for example, in GenBank Accession NumberAF226675), SOX 10 (as described, for example, in GenBank AccessionNumber AJ001183), SOX 12 (as described, for example, in GenBankAccession Number X73039) and SOX 21 (as described, for example, inGenBank Accession Number AF107044)); lymphoid specific factor (LEF-1)(as described, for example, in GenBank Accession Number X58636); T-cellspecific transcription factor (TCF-1) (as described, for example, inGenBank Accession Number X59869); MTT1 (as described, for example, inGenBank Accession Number M62810); and SP100-HMG nuclear autoantigen (asdescribed, for example, in GenBank Accession Number U36501).

Other examples of HMGB proteins are polypeptides encoded by HMGB nucleicacid sequences having GenBank Accession Numbers NG_(—)000897 (HMG1L10)(and in particular by nucleotides 658-1305 of NG_(—)000897, as shown inFIGS. 7A and 7B); AF076674 (HMG1L1) (and in particular by nucleotides1-633 of AF076674, as shown in FIGS. 7C and 7D; AF076676 (HMG1L4 ) (andin particular by nucleotides 1-564 of AF076676, as shown in FIGS. 7E and7F); AC010149 (HMG sequence from BAC clone RP11-395A23) (and inparticular by nucleotides 75503-76117 of AC010149), as shown in FIGS. 7Gand 7H); AF165168 (HMG1L9) (and in particular by nucleotides 729-968 ofAF165168, as shown in FIGS. 7I and 7J); XM_(—)063129 (LOC122441) (and inparticular by nucleotides 319-558 of XM_(—)063129, as shown in FIGS. 7Kand 7L); XM_(—)066789 (LOC139603) (and in particular by nucleotides1-258 of XM_(—)066789, as shown in FIGS. 7M and 7N); and AF165167(HMG1L8) (and in particular by nucleotides 456-666 of AF165167, as shownin FIGS. 7O and 7P).

Optionally, the HMGB polypeptide is a substantially pure, orsubstantially pure and isolated polypeptide that has been separated fromcomponents that naturally accompany it. As used herein, a polypeptide issaid to be “isolated” or “purified” when it is substantially free ofcellular material when it is isolated from recombinant andnon-recombinant cells, or free of chemical precursors or other chemicalswhen it is chemically synthesized. A polypeptide, however, can be joinedto another polypeptide with which it is not normally associated in acell (e.g., in a “fusion protein”) and still be “isolated” or“purified.” It is understood, however, that preparations in which thepolypeptide is not purified to homogeneity are useful. For example, thepolypeptide may be in an unpurified form, for example, in a cell, cellmilieu, or cell extract. The critical feature is that the preparationallows for the desired function of the polypeptide, even in the presenceof considerable amounts of other components.

Functional equivalents of HMGB (proteins or polypeptides that have oneor more of the biological activities of an HMGB polypeptide) can also beused in the methods of the present invention. Biologically activefragments, sequence variants, post-translational modifications, andchimeric or fusion proteins comprising the protein, biologically activefragment, or variant are examples of functional equivalents of aprotein. Variants include a substantially homologous polypeptide encodedby the same genetic locus in an organism, i.e., an allelic variant, aswell as other splicing variants. Variants also encompass polypeptidesderived from other genetic loci in an organism, but having substantialhomology to the protein of interest, for example, an HMGB protein asdescribed herein.

A variant polypeptide can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these. Further, variantpolypeptides can be fully functional or can lack function in one or moreactivities. Fully functional variants typically contain onlyconservative variation or variation in non-critical residues or innon-critical regions. Functional variants can also contain substitutionof similar amino acids that result in no change or an insignificantchange in function. Alternatively, such substitutions may positively ornegatively affect function to some degree. Non-functional variantstypically contain one or more non-conservative amino acid substitutions,deletions, insertions, inversions, or truncation or a substitution,insertion, inversion, or deletion in a critical residue or criticalregion.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science, 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity in vitro. Sites that are critical for polypeptideactivity can also be determined by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith et al., J. Mol. Biol., 224:899-904 (1992); de Vos et al.,Science, 255:306-312 (1992)).

HMGB functional equivalents also encompass polypeptides having a lowerdegree of identity but having sufficient similarity so as to perform oneor more of the same functions performed by an HMGB polypeptide.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Conservativesubstitutions are likely to be phenotypically silent. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu and Ile; interchange of thehydroxyl residues Ser and Thr, exchange of the acidic residues Asp andGlu, substitution between the amide residues Asn and Gln, exchange ofthe basic residues Lys and Arg and replacements among the aromaticresidues Phe and Tyr. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science,247:1306-1310 (1990).

HMGB functional equivalents also include polypeptide fragments of HMGB.Fragments can be derived from an HMGB polypeptide or fragments of HMGBvariants. As used herein, a fragment comprises at least 6 contiguousamino acids. Useful fragments include those that retain one or more ofthe biological activities of the polypeptide. Examples of HMGBbiologically active fragments include the B box, as well as fragments ofthe B box, for example, the first 20 amino acids of the B box (e.g. thefirst 20 amino acids of SEQ ID NOs: 5 (SEQ ID NO: 44) or 8 (SEQ ID NO:45)).

Biologically active fragments (peptides which are, for example, 6, 9,12, 15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acidsin length) can comprise a domain, segment, or motif that has beenidentified by analysis of the polypeptide sequence using well-knownmethods, e.g., signal peptides, extracellular domains, one or moretransmembrane segments or loops, ligand binding regions, zinc fingerdomains, DNA binding domains, or post-translation modification sites.Example of domains include the A box and/or the B box as describedherein.

Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the polypeptide fragment and an additional region fused tothe carboxyl terminus of the fragment.

The invention also provides uses and methods for chimeric or fusionpolypeptides containing an HMGB polypeptide, biologically activefragment thereof, or variant thereof as functional equivalents of HMGB.These chimeric proteins comprise an HMGB polypeptide or functionalequivalent thereof operatively linked to a heterologous protein orpolypeptide having an amino acid sequence not substantially homologousto the polypeptide. “Operatively linked” indicates that the polypeptideand the heterologous protein are fused in-frame. The heterologousprotein can be fused to the N-terminus or C-terminus of the polypeptide.In one embodiment the fusion polypeptide does not affect function of thepolypeptide per se. For example, the fusion polypeptide can be aGST-fusion polypeptide in which the polypeptide sequences are fused tothe C-terminus of the GST sequences. Other types of fusion polypeptidesinclude, but are not limited to, enzymatic fusion polypeptides, forexample, β-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, FLAG-tagged fusions, GFP fusions, and Ig fusions. Such fusionpolypeptides can facilitate the purification of recombinant polypeptide.In certain host cells (e.g., mammalian host cells) expression and/orsecretion of a polypeptide can be increased by using a heterologoussignal sequence. Therefore, in another embodiment, the fusionpolypeptide contains a heterologous signal sequence at its N-terminus.

EP-A-O 464 533 discloses fusion proteins comprising various portions ofimmunoglobulin constant regions. The Fc is useful in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists. Bennett etal., Journal of Molecular Recognition 8:52-58 (1995) and Johanson etal., The Journal of Biological Chemistry, 270(16):9459-9471 (1995).Thus, this invention also encompasses soluble fusion polypeptidescontaining a polypeptide of the invention and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclass (IgG, IgM, IgA, IgE).

A chimeric or fusion polypeptide can be produced by standard recombinantDNA techniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of nucleic acid fragmentscan be carried out using anchor primers that give rise to complementaryoverhangs between two consecutive nucleic acid fragments that cansubsequently be annealed and re-amplified to generate a chimeric nucleicacid sequence (see Ausubel et al., Current Protocols in MolecularBiology, 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST protein). Anucleic acid molecule encoding an HMGB polypeptide can be cloned intosuch an expression vector such that the fusion moiety is linked in-frameto the polypeptide.

HMGB functional equivalents can be generated using standard molecularbiology techniques and assaying the function using, for example, methodsdescribed herein, such as, determining if the functional equivalent,when administered to a cell (e.g., a macrophage) increases release of aproinflammatory cytokine from the cell, compared to an untreated controlcell.

HMGB polypeptides can be purified from cells that naturally express it,purified from cells that have been altered to express it (recombinant),or synthesized using known protein synthesis methods. In one embodiment,the polypeptide is produced by recombinant DNA techniques. For example,a nucleic acid molecule encoding the polypeptide is cloned into anexpression vector, the expression vector introduced into a host cell andthe polypeptide expressed in the host cell. The polypeptide can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques.

As used herein, an “HMGB A box” also referred to herein as an “A box”(and also known as HMG A box) is a protein or polypeptide that has oneor more of the following biological activities: inhibiting inflammationmediated by HMGB and/or inhibiting release of a proinflammatory cytokinefrom a cell. In one embodiment, the HMGB A box polypeptide has one ofthe above biological activities. Typically, the HMGB A box polypeptidehas both of the above biological activities. In one embodiment, the Abox has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequenceidentity to SEQ ID NOs: 4 or 7. Preferably, the HMGB A box has no morethan 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of thebiological activity of full length HMGB. In one embodiment, the HMGB Abox amino acid consists of the sequence of SEQ ID NO: 4 or SEQ ID NO: 7(PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGET) or the amino acid sequence in thecorresponding region of an HMGB protein in a mammal. An HMGB A box isalso a recombinantly produced polypeptide having the same amino acidsequence as the A box sequences described above. Preferably, the HMGB Abox is a mammalian HMGB A box, for example, a human HMGB1 A box.

An HMGB A box often has no more than about 85 amino acids and no fewerthan about 4 amino acids. Examples of polypeptides having A boxsequences within them include, but are not limited to HMGB polypeptidesdescribed herein; GenBank Accession Numbers AAA64970, AAB08987, P07155,AAA20508, S29857, P09429, NP_(—)002119, CAA31110, S02826, U00431,X67668, NP_(—)005333, NM_(—)016957, and J04197, mammalian HMG1 ((HMGB1)as described, for example, in GenBank Accession Number U51677), HMG2((HMGB2) as described, for example, in GenBank Accession Number M83665),HMG-2A ((HMGB3, HMG-4) as described, for example, in GenBank AccessionNumbers NM_(—)005342 and NP_(—)005333), HMG14 (as described, forexample, in GenBank Accession Number P05114), HMG17 (as described, forexample, in GenBank Accession Number X13546), HMGI (as described, forexample, in GenBank Accession Number L17131), and HMGY (as described,for example, in GenBank Accession Number M23618); nonmammalian HMG T1(as described, for example, in GenBank Accession Number X02666) and HMGT2 (as described, for example, in GenBank Accession Number L32859)(rainbow trout); HMG-X (as described, for example, in GenBank AccessionNumber D30765) (Xenopus), HMG D (as described, for example, in GenBankAccession Number X71138) and HMG Z (as described, for example, inGenBank Accession Number X71139) (Drosophila); NHP10 protein (HMGprotein homolog NHP 1) (as described, for example, in GenBank AccessionNumber Z48008) (yeast); non-histone chromosomal protein (as described,for example, in GenBank Accession Number O00479) (yeast); HMG 1/2 likeprotein (as described, for example, in GenBank Accession Number Z11540)(wheat, maize, soybean); upstream binding factor (UBF-1) (as described,for example, in GenBank Accession Number X53390); PMS1 protein homolog 1(as described, for example, in GenBank Accession Number U13695);single-strand recognition protein (SSRP, structure-specific recognitionprotein) (as described, for example, in GenBank Accession NumberM86737); the HMG homolog TDP-1 (as described, for example, in GenBankAccession Number M74017); mammalian sex-determining region Y protein(SRY, testis-determining factor) (as described, for example, in GenBankAccession Number X53772); fungal proteins: mat-1 (as described, forexample, in GenBank Accession Number AB00945 1), ste 11 (as described,for example, in GenBank Accession Number x53431) and Mc 1; SOX 14 (asdescribed, for example, in GenBank Accession Number AF107043) (as wellas SOX 1 (as described, for example, in GenBank Accession NumberY13436), SOX 2 (as described, for example, in GenBank Accession NumberZ31560), SOX 3 (as described, for example, in GenBank Accession NumberX71135), SOX 6 (as described, for example, in GenBank Accession NumberAF309034), SOX 8 (as described, for example, in GenBank Accession NumberAF226675), SOX 10 (as described, for example, in GenBank AccessionNumber AJ001183), SOX 12 (as described, for example, in GenBankAccession Number X73039) and SOX 21 (as described, for example, inGenBank Accession Number AF107044)); lymphoid specific factor (LEF-1)(as described, for example, in GenBank Accession Number X58636); T-cellspecific transcription factor (TCF-1) (as described, for example, inGenBank Accession Number X59869); MTT1 (as described, for example, inGenBank Accession Number M62810) and SP 100-HMG nuclear autoantigen (asdescribed, for example, in GenBank Accession Number U36501).

Other examples of polypeptides having A box sequences within theminclude, but are not limited to polypeptides encoded by GenBankAccession Numbers NG_(—)000897 (HMG1L10) (and in particular bynucleotides 658-1305 of NG_(—)000897, as shown in FIGS. 7A and 7B);AF076674 (HMG1L1) (and in particular by nucleotides 1-633 of AF076674,as shown in FIGS. 7C and 7D; AF076676 (HMG1L4 ) (and in particular bynucleotides 1-564 of AF076676, as shown in FIGS. 7E and 7F); AC010149(HMG sequence from BAC clone RP11-395A23) (and in particular bynucleotides 75503-76117 of AC010149), as shown in FIGS. 7G and 7H);AF165168 (HMG1L9) (and in particular by nucleotides 729-968 of AF165168,as shown in FIGS. 7I and 7J); XM_(—)063129 (LOC122441) (and inparticular by nucleotides 319-558 of XM_(—)063129, as shown in FIGS. 7Kand 7L); XM_(—)066789 (LOC139603) (and in particular by nucleotides1-258 of XM_(—)066789, as shown in FIGS. 7M and 7N); and AF165167(HMG1L8) (and in particular by nucleotides 456-666 of AF165167, as shownin FIGS. 7O and 7P). The A box sequences in such polypeptides can bedetermined and isolated using methods described herein, for example, bysequence comparisons to A boxes described herein and testing forbiological activity using methods described herein or other method knownin the art.

Additional examples of HMGB A box polypeptide sequences include thefollowing sequences: PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARYEREMKTYIPP KGET (human HMGB1; SEQ ID NO: 25); DSSVNFAEF SKKCSERWKTMSAKEKSKFE DMAKSDKARY DREMKNYVPP KGDK (human HMGB2; SEQ ID NO: 26);PEVPVNFAEF SKKCSERWKT VSGKEKSKFD EMAKADKVRY DREMKDYGPA KGGK (humanHMGB3; SEQ ID NO: 27); PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARYEREMKTYIPP KGET (HMG1L10; SEQ ID NO: 28); SDASVNFSEF SNKCSERWKTMSAKEKGKFE DMAKADKTHY ERQMKTYIPP KGET (HMG1L1; SEQ ID NO: 29);PDASVNFSEF SKKCSERWKA MSAKDKGKFE DMAKVDKADY EREMKTYIPP KGET (HMG1L4; SEQID NO: 30); PDASVKFSEF LKKCSETWKT IFAKEKGKFE DMAKADKAHY EREMKTYIPP KGEK(HMG sequence from BAC clone RP11-395A23; SEQ ID NO: 31); PDASINFSEFSQKCPETWKT TIAKEKGKFE DMAKADKAHY EREMKTYIPP KGET (HMG1L9; SEQ ID NO:32); PDASVNSSEF SKKCSERWKTMPTKQGKFE DMAKADRAH (HMG1L8; SEQ ID NO: 33);PDASVNFSEF SKKCLVRGKT MSAKEKGQFE AMARADKARY EREMKTYIP PKGET (LOC122441;SEQ ID NO: 34); LDASVSFSEF SNKCSERWKT MSVKEKGKFE DMAKADKACY EREMKIYPYLKGRQ (LOC139603; SEQ ID NO: 35); and GKGDPKKPRG KMSSYAFFVQ TCREEHKKKHPDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (human HMGB1A box; SEQ ID NO: 36).

Functional equivalents of HMGB A boxes can also be used to carry out themethods of the present invention. Examples of HMGB A box functionalequivalents include, for example, biologically active fragments,post-translational modifications, variants, or fusion proteinscomprising A boxes, as defined herein. A box functional equivalents canbe generated using standard molecular biology techniques and assayingthe function using known methods, for example, by determining if thefragment, when administered to a cell (e.g., a macrophage) decreases orinhibits release of a proinflammatory cytokine from the cell.

Optionally, the HMGB A box polypeptide is a substantially pure, orsubstantially pure and isolated polypeptide that has been separated fromcomponents that naturally accompany it. Alternatively, the polypeptidemay be in an unpurified form, for example, in a cell, cell milieu, orcell extract. The critical feature is that the preparation allows forthe desired function of the polypeptide, even in the presence ofconsiderable amounts of other components.

As used herein, an “HMGB B box” also referred to herein as a “B box”(and also known as an HMG B box) is a polypeptide that has at least 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NOs: 5or 8 (as determined using the BLAST program and parameters describedherein), lacks an A box, and has one or more of the following biologicalactivities: increasing inflammation and/or increasing release of aproinflammatory cytokine from a cell. In one embodiment, the HMGB B boxpolypeptide has one of the above biological activities. Typically, theHMGB B box polypeptide has both of the above biological activities.Preferably, the HMGB B box has at least 25%, 30%, 40%, 50%, 60%, 70%,80% or 90% of the biological activity of full length HMGB. In anotherembodiment, the HMGB box comprises or consists of the sequence of SEQ IDNO: 5 or SEQ ID NO: 8(FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAY) or the amino acid sequence in the correspondingregion of an HMGB protein in a mammal.

Preferably, the HMGB B box is a mammalian HMGB B box, for example, ahuman HMGB1 B box. An HMGB B box often has no more than about 85 aminoacids and no fewer than about 4 amino acids. Examples of polypeptideshaving B box sequences within them include, but are not limited to HMGBpolypeptides described herein; GenBank Accession Numbers AAA64970,AAB08987, P07155, AAA20508, S29857, P09429, NP_(—)002119, CAA31110,S02826, U00431, X67668, NP_(—)005333, NM_(—)016957, and J04197,mammalian HMG1 ((HMGB1) as described, for example, in GenBank AccessionNumber U51677), HMG2 ((HMGB2) as described, for example, in GenBankAccession Number M83665), HMG-2A ((HMGB3, HMG-4) as described, forexample, in GenBank Accession Numbers NM_(—)005342 and NP_(—)005333),HMG14 (as described, for example, in GenBank Accession Number P05114),HMG 17 (as described, for example, in GenBank Accession Number X13546),HMGI (as described, for example, in GenBank Accession Number L17131),and HMGY (as described, for example, in GenBank Accession NumberM23618); nonmammalian HMG T1 (as described, for example, in GenBankAccession Number X02666) and HMG T2 (as described, for example, inGenBank Accession Number L32859) (rainbow trout); HMG-X (as described,for example, in GenBank Accession Number D30765) (Xenopus), HMG D (asdescribed, for example, in GenBank Accession Number X71138) and HMG Z(as described, for example, in GenBank Accession Number X71139)(Drosophila); NHP10 protein (HMG protein homolog NHP 1) (as described,for example, in GenBank Accession Number Z48008) (yeast); non-histonechromosomal protein (as described, for example, in GenBank AccessionNumber 000479) (yeast); HMG 1/2 like protein (as described, for example,in GenBank Accession Number Z11540) (wheat, maize, soybean); upstreambinding factor (UBF-1) (as described, for example, in GenBank AccessionNumber X53390); PMS1 protein homolog 1 (as described, for example, inGenBank Accession Number U13695); single-strand recognition protein(SSRP, structure-specific recognition protein) (as described, forexample, in GenBank Accession Number M86737); the HMG homolog TDP-1 (asdescribed, for example, in GenBank Accession Number M74017); mammaliansex-determining region Y protein (SRY, testis-determining factor) (asdescribed, for example, in GenBank Accession Number X53772); fungalproteins: mat-1 (as described, for example, in GenBank Accession NumberAB00945 1), ste 11 (as described, for example, in GenBank AccessionNumber x53431) and Mc 1; SOX 14 (as described, for example, in GenBankAccession Number AF107043) (as well as SOX 1 (as described, for example,in GenBank Accession Number Y13436), SOX 2 (as described, for example,in GenBank Accession Number Z31560), SOX 3 (as described, for example,in GenBank Accession Number X71135), SOX 6 (as described, for example,in GenBank Accession Number AF309034), SOX 8 (as described, for example,in GenBank Accession Number AF226675), SOX 10 (as described, forexample, in GenBank Accession Number AJ001 183), SOX 12 (as described,for example, in GenBank Accession Number X73039) and SOX 21 (asdescribed, for example, in GenBank Accession Number AF107044)); lymphoidspecific factor (LEF-1) (as described, for example, in GenBank AccessionNumber X58636); T-cell specific transcription factor (TCF-1) (asdescribed, for example, in GenBank Accession Number X59869); MTT1 (asdescribed, for example, in GenBank Accession Number M62810); andSP100-HMGB nuclear autoantigen (as described, for example, in GenBankAccession Number U36501).

Other examples of polypeptides having B box sequences within theminclude, but are not limited to polypeptides encoded by GenBankAccession Numbers NG_(—)000897 (HMG1L10) (and in particular bynucleotides 658-1305 of NG_(—)000897, as shown in FIGS. 7A and 7B);AF076674 (HMG1L1) (and in particular by nucleotides 1-633 of AF076674,as shown in FIGS. 7C and 7D; AF076676 (HMG1L4 ) (and in particular bynucleotides 1-564 of AF076676, as shown in FIGS. 7E and 7F); AC010149(HMG sequence from BAC clone RP11-395A23) (and in particular bynucleotides 75503-76117 of AC010149), as shown in FIGS. 7G and 7H) The Bbox sequences in such polypeptides can be determined and isolated usingmethods described herein, for example, by sequence comparisons to Bboxes described herein and testing for biological activity.

Examples of HMGB B box polypeptide sequences include the followingsequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAADDKQPYEKKA AKLKEKYEKD IAAY (human HMGB1; SEQ ID NO: 37); KKDPNAPKRPPSAFFLFCSE HRPKIKSEHP GLSIGDTAKK LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY(human HMGB2; SEQ ID NO: 38); FKDPNAPKRL PSAFFLFCSE YRPKIKGEHPGLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (HMG1L10; SEQ ID NO:39); FKDPNAPKRP PSAFFLFCSE YHPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPGEKKAAKLKEKYEKD IAAY (HMG1L1; SEQ ID NO: 40); FKDSNAPKRP PSAFLLFCSEYCPKIKGEHP GLPISDVAKK LVEMWNNTFA DDKQLCEKKA AKLKEKYKKD TATY (HMG1L4; SEQID NO: 41); FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVVKK LAGMWNNTAAADKQFYEKKA AKLKEKYKKD IAAY (HMG sequence from BAC clone RP11-359A23; SEQID NO: 42); and FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAADDKQPYEKKA AKLKEKYEKD IAAYRAKGKP DAAKKGVVKA EK (human HMGB1 box; SEQ IDNO: 43).

Functional equivalents of HMGB B boxes can also be used to carry out themethods of the present invention. Examples of HMGB B box functionalequivalents include, for example, biologically active fragments,post-translational modifications, variants, or fusion proteinscomprising B boxes, as defined herein. B box functional equivalents canbe generated using standard molecular biology techniques and assayingthe function using known methods, for example, by determining if thefragment, when administered to a cell (e.g., a macrophage) increasesrelease of a proinflammatory cytokine from the cell.

Optionally, the HMGB B box polypeptide is a substantially pure, orsubstantially pure and isolated polypeptide that has been separated fromcomponents that naturally accompany it. Alternatively, the polypeptidemay be in an unpurified form, for example, in a cell, cell milieu, orcell extract. The critical feature is that the preparation allows forthe desired function of the polypeptide, even in the presence ofconsiderable amounts of other components.

HMGB polypeptides, HMGB A boxes, and HMGB B boxes, either naturallyoccurring or non-naturally occurring, include polypeptides that havesequence identity to the HMGB, HMGB A boxes, and HMGB B boxes describedabove. As used herein, two polypeptides (or a region of thepolypeptides) are substantially homologous or identical when the aminoacid sequences are at least about 60%, 70%, 75%, 80%, 85%, 90% or 95% ormore homologous or identical. The percent identity of two amino acidsequences (or two nucleic acid sequences) can be determined by aligningthe sequences for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first sequence). The amino acids ornucleotides at corresponding positions are then compared, and thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions×100). In certain embodiments,the length of the polypeptide aligned for comparison purposes is atleast 30%, preferably, at least 40%, more preferably, at least 60%, andeven more preferably, at least 70%, 80%, 90%, or 100% of the length ofthe reference sequence, for example, those sequences provided herein.The actual comparison of the two sequences can be accomplished bywell-known methods, for example, using a mathematical algorithm. Apreferred, non-limiting example of such a mathematical algorithm isdescribed in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993). Such an algorithm is incorporated into the BLASTN and BLASTXprograms (version 2.2) as described in Schaffer et al., Nucleic AcidsRes., 29:2994-3005 (2001). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,BLASTN) can be used. See, for example, the National Center forBiotechnology Information database. In one embodiment, the databasesearched is a non-redundant (NR) database, and parameters for sequencecomparison can be set at: no filters; Expect value of 10; Word Size of3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and anExtension of 1.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0), which is part of the GCG (Accelrys) sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12 , and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl.Biosci., 10:3-5 (1994); and FASTA described in Pearson and Lipman, Proc.Natl. Acad. Sci. USA, 85:2444-8 (1988).

In another embodiment, the percent identity between two amino acidsequences can be accomplished using the GAP program in the GCG softwarepackage (Accelrys, Inc., San Diego, Calif.) using either a Blossom 63matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and alength weight of 2, 3, or 4. In yet another embodiment, the percentidentity between two nucleic acid sequences can be accomplished usingthe GAP program in the GCG software package (Accelrys, San Diego,Calif.), using a gap weight of 50 and a length weight of 3.

As used herein, a Toll-like receptor 2 (TLR2) protein is a polypeptidethat can bind an HMGB protein and mediate inflammation and/or release ofa pro-inflammatory cytokine from a cell and has at least 50%, 60%, 70%,75%, 80%, 85%, 90% or 95% sequence identity of SEQ ID NO: 46 (GenBankAccession Number AAC34133). In one embodiment the TLR2 is mammalian, forexample, human. In another embodiment, the TLR2 comprises or consists ofthe amino acid sequence of SEQ ID NO: 46. In still another embodiment,the TLR2 protein is encoded by the nucleic acid sequence of SEQ ID NO:47 (GenBank Accession number U88878). Optionally, the TLR2 protein is asubstantially pure, or substantially pure and isolated polypeptide thathas been separated from components that naturally accompany it, or arecombinantly produced polypeptide having the same amino acid sequence.Alternatively, the TLR2 protein may be in an unpurified form, forexample, in a cell (intracellular), a plasma membrane (on the surface ofa cell or in a vesicle (e.g., an endosome), or a cell extract. TLR2proteins also include functional equivalents of TLR2, for example,biologically active fragments, post-translational modifications, orvariants, as defined herein.

As used herein, a “cytokine” is a protein or peptide that is naturallyproduced by mammalian cells and that acts in vivo as a regulator atmicro- to picomolar concentrations. Cytokines can, either under normalor pathological conditions, modulate the functional activities ofindividual cells and tissues. A proinflammatory cytokine is a cytokinethat is capable of causing (directly or indirectly) a physiologicalreaction associated with inflammation, for example, vasodilation,hyperemia, increased permeability of vessels with associated edema,accumulation of granulocytes and mononuclear phagocytes, or depositionof fibrin. In some cases, the proinflammatory cytokine can also causeapoptosis, such as in chronic heart failure, where TNF has been shown tostimulate cardiomyocyte apoptosis (Pulkki, Ann. Med. 29:339-343 (1997);and Tsutsui et al., Immunol. Rev. 174:192-209 (2000)).

Nonlimiting examples of proinflammatory cytokines are tumor necrosisfactor (TNF), interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-18, interferonγ, HMG-1 (also know as HMG1, and HMGB1), and macrophage migrationinhibitory factor (MIF).

Proinflammatory cytokines are to be distinguished from anti-inflammatorycytokines, such as IL-4, IL-10, and IL-13, which are not mediators ofinflammation.

In many instances, proinflammatory cytokines are produced in aninflammatory cytokine cascade, defined herein as an in vivo release ofat least one proinflammatory cytokine in a mammal, wherein the cytokinerelease affects a physiological condition of the mammal. Thus, aninflammatory cytokine cascade is inhibited in embodiments of theinvention where proinflammatory cytokine release causes a deleteriousphysiological condition.

When referring to the effect of any of the compositions or methods ofthe invention on the release of proinflammatory cytokines, the use ofthe terms “inhibit” or “decrease” encompasses at least a small butmeasurable reduction in proinflammatory cytokine release. In preferredembodiments, the release of the proinflammatory cytokine is inhibited byat least 20% over non-treated controls; in more preferred embodiments,the inhibition is at least 50%; in still more preferred embodiments, theinhibition is at least 70%, and in the most preferred embodiments, theinhibition is at least 80%. Such reductions in proinflammatory cytokinerelease are capable of reducing the deleterious effects of aninflammatory cytokine cascade in in vivo embodiments.

Agents that Inhibit the Interaction Between HMGB and TLR2

The present invention provides screening methods for identifying agents(inhibitors) that inhibit the interaction (e.g., binding) between HMGBand TLR2. The agents can be used in therapeutic methods as describedherein. As used herein, an “inhibitor” is an agent that acts byinhibiting (preventing or decreasing) at least one functioncharacteristic of the interaction between an HMGB protein and a TLR2,such as a binding activity (complex formation), cellular signalingtriggered by the interaction and/or cellular response function (e.g., aninflammatory response, release of a proinflammatory cytokine from acell) mediated by the interaction.

As used herein, the term inhibitor includes agents that bind to TLR2 andprevents binding thereto by HMGB. In one embodiment the agent binds TLR2(e.g., an antibody or antigen-binding fragment, a mutant of a naturalligand, a peptidomimetic, and other competitive inhibitors of ligandbinding) and inhibits binding by HMGB. In one embodiment, the agent is aligand that binds to TLR2 with greater affinity than HMGB binds to TLR2.Preferably the agent that binds to TLR2, thereby inhibiting binding byHMGB, does not significantly initiate or increase an inflammatoryresponse, or does not significantly initiate or increase the release ofa proinflammatory cytokine from a cell.

Examples of ligands that are known to bind TLR2 include: heat shockprotein 60, surfactant protein-A, monophosphoryl lipid A (Botler et al.,Infect. Immun. 71(5):2498-2507 (2003)), muramyl dipeptide (Beutler etal., Blood Cells Mol. Dis. 27(4):728-730 (2001)), yeast-particlezymosan, GPI anchor from Trypanosoma cruzi, Listeria monocytogenes,Bacillus, lipoteichoic acid, peptidoglycan, and lipopeptides fromStreptococcus species, heat killed Mycobacteriua tuberculosis,Mycobacteria avium lipopeptide, lipoarabinomannan, mannosylatedphosphatidylinositol, Borrelia burgdorferi, Treponema pallidum,Treponema maltophilum (lipopeptides, glycolipids, outer surface proteinA), and MALP-2 lipopeptides from Mycoplasma fermentans. Therefore, thesemolecules, as well as portions of these molecules that bind TLR2 can beused to inhibit the interaction between HMGB and TLR2. It is alsoreasonable to believe that another ligand for TLR2 is the HMGB A box andTLR2 binding fragments thereof (see, for example, U.S. application Ser.No. 10/147,447, the entire teaching of which are incorporated herein byreference).

In another embodiment, the inhibitor binds HMGB, and prevents HMGB frombinding to TLR2. Such an agent can be, for example, a soluble form ofrecombinant TLR2 (sTLR2) (i.e., TLR2 lacking the intracellular andtransmembrane domains, as described, for example, by Iwaki et al.,Journal of Biological Chemistry 277(27):24315-24320 (2002)) or annon-HMG1 antibody molecule (e.g., a protein, peptide, or non-peptidicsmall molecule) that binds HMG1 and prevents it from binding to TLR2.The sTLR2 molecule can contain the extracellular domain (for example,amino acids 1-587 of the TLR2 amino acid sequence (e.g., GenBankAccession Number). The sTLR molecule can also be modified with one ofmore amino acid substitutions and/or post-translational modificationsprovided such sTLR2 molecules have HMGB binding activity, which can beassessed using methods known in the art. Such sTLR2 molecules can bemade, for example, using recombinant techniques. Preferably the sTLR2has at least 70%, 75%, 80%, 85%, 90%, or 95% to amino acids 1-587 ofGenBank Accession Number AAC34133. In another embodiment, the inhibitoris an agent that bind TLR2 a site different than the HMGB binding siteand blocks binding by HMGB (e.g., by causing a conformation change inthe TLR2 protein or otherwise altering the binding site for HMGB).

In one embodiment, the inhibitor is not an anti-TLR2 antibody orantigen-binding fragment thereof. In another embodiment, the inhibitoris not an antibody that binds HMGB1 (an anti-HMGB1 antibody) or anantigen-binding fragment thereof. In another embodiment, the inhibitoris not an antibody that binds HMGB (an anti-HMGB antibody) or anantigen-binding fragment thereof. In another embodiment, the inhibitoris not soluble RAGE (i. e., a portion of the RAGE receptor that bindsHMGB1). In another embodiment, the inhibitor is non-microbial (i.e., isnot a microbe, derived from a microbe, or secreted or released from amicrobe). In still another embodiment, the inhibitor is a mammalianinhibitor (i.e., is a molecule that naturally exists in a mammal, isderived from a molecule that naturally exists in a mammal, or issecreted or released from a mammalian cell), for example, a humaninhibitor. Preferably, the inhibitor inhibits the interaction betweenHMGB and TLR2 by at least about 10%, 25%, 50%, 60%, 70%, 80%, 90% or100% compared to a suitable control (e.g., a sample receiving noinhibitor or receiving the inhibitor vehicle only). In anotherembodiment, the inhibitor is a small molecule inhibitor (i.e., having amolecular weight of 1000 or less, 500 or less, 250 or less or 100 orless). In another embodiment the inhibitor is a short peptide, having,for example, 50 or fewer amino acids, 30 or fewer amino acids, 25 orfewer amino acids, 20 or fewer amino acids, 10 or fewer amino acids, or5 or fewer amino acids. In another embodiment, the inhibitor is not anHMGB A box. In another embodiment the inhibitor is not a peptide. Inanother embodiment, the inhibitor inhibits the interaction between HMGBand TLR2 at a level useful in vivo, such that the dose can be toleratedby an individual and would be useful to an individual. Preferably theactivity of the inhibitor is such that less than 1 g, less than 100 mg,less than 10 mg, less than 1 mg, less than 100 μg, less than 10 μg, lessthan 1 μg, less than 100 ng, less than 10 ng, or less than 1 ng of theinhibitor is used in the assays and/or therapeutic methods describedherein or comparable assays or methods.

Antibodies

In one embodiment, the inhibitor is an antibody or an antigen-bindingfragment of an antibody. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that selectively binds an antigen (e.g., an antigen-bindingfragment of an antibody). The antibodies of the present invention aremolecules that selectively bind to a TLR2 or functional equivalentthereof (e.g., a fragment thereof). In a preferred embodiment, theantibodies do not substantially bind other molecules in a sample.Preferably, the antibody is at least 60%, by weight, free from proteinsand naturally occurring organic molecules with which it naturallyassociated. More preferably, the antibody preparation is at least 75% or90%, and most preferably, 99%, by weight, antibody.

The antibodies of the present invention can be polyclonal or monoclonal,and the term antibody is intended to encompass both polyclonal andmonoclonal antibodies. The terms polyclonal and monoclonal refer to thedegree of homogeneity of an antibody preparation, and are not intendedto be limited to particular methods of production.

Antibodies of the present invention can be raised against an appropriateimmunogen, including proteins or polypeptides of TLR2 described herein(including synthetic molecules, such as synthetic peptides).

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256:495-497 (1975)) and Eur. J. Immunol. 6:511-519 (1976)); Milstein etal., Nature 266:550-552 (1977)); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); andCurrent Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, 1991); the teachings of each of which are incorporatedherein by reference). Generally, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cell, preferablythose of the spleen or lymph nodes, can be obtained from animalsimmunized with the antigen of interest. The fused cells (hybridomas) canbe isolated using selective culture conditions, and cloned by limitingdilution. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can used, including, for example, methods whichselect recombinant antibody from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies (see e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-2555 (1993)); Jakobovits et al., Nature,362:255-258 (1993)); Lonberg et al., U.S. Pat. No. 5,545,806; and Suraniet al., U.S. Pat. No. 5,545,807; the teachings of which are eachincorporated herein by reference).

Single-chain antibodies, and chimeric, humanized or primatized(CDR-grafted), or veneered antibodies, as well as chimeric, CDR-graftedor veneered single-chain antibodies, comprising portions derived fromdifferent species, and the like are also encompassed by the presentinvention and the term “antibody.” The various portions of theseantibodies can be joined together chemically by conventional techniques,or can be prepared as a contiguous protein using genetic engineeringtechniques. For example, nucleic acids encoding a chimeric or humanizedchain can be expressed to produce a contiguous protein. See, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen etal., European Patent No. 0 451 216 B1; and Padlan et al., EP 0 519 596A1. See also, Newman et al., BioTechnology, 10:1455-1460 (1992),regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird et al., Science, 242:423-426 (1988) regardingsingle-chain antibodies.

In addition, antigen-binding fragments of antibodies, includingfragments of chimeric, humanized, primatized, veneered or single-chainantibodies, can also be produced. In one embodiment, the antigen-bindingfragment is a soluble TLR2 molecule (e.g., an extracellular TLR2 domainas described, for example, by Iwaki et al., supra). Antigen-bindingfragments of foregoing antibodies retain at least one binding functionand/or modulation function of the full-length antibody from which theyare derived. For example, antigen-binding fragments capable of bindingto a TLR2 or a functional variant thereof, including, but not limitedto, Fv, Fab, Fab′ and F(ab′)₂ fragments are encompassed by theinvention. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Reduction of thedisulfide bond between the heavy chains of F(ab′)₂ fragments can yieldF(ab′) fragments. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain portion can be designed toinclude DNA sequences encoding the CH, domain and hinge region of theheavy chain.

The antibodies and fragments of the present invention can be modified,for example, by incorporation of or attachment (directly or indirectly)of a detectable label. Examples of detectable labels include variousspin labels, antigen or epitope tags, haptens, enzyme labels, prostheticgroups, fluorescent materials, chemiluminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzyme labels include horseradish peroxidase, alkalinephosphatase, β-galactosidase, and acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidinibiotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;an example of a chemiluminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,and ³H.

Aptamers

In one embodiment, the agent that inhibits interaction between HMGB andTLR2 is an aptamer. Aptamers are single stranded oligonucleotides oroligonucleotide analogs that bind to a particular target molecule, suchas a protein or a small molecule (e.g., a steroid or a drug, etc.).Thus, aptamers are the oligonucleotide analogy to antibodies. However,aptamers are smaller than antibodies, generally in the range of 50-100nucleotides. Their binding is highly dependent on the secondarystructure formed by the aptamer oligonucleotide. Both RNA and singlestranded DNA (or analog), aptamers are known. See, e.g., Burke et al.,J. Mol. Biol. 264:650 (1996); Ellington and Szostak, Nature 346:818(1990); Hirao et al., Mol. Divers. 4:75 (1998); Jaeger et al., The EMBOJournal 17:4535 (1998); Kensch et al., J. Biol. Chem. 275:18271 (2000);Schneider et al., Biochemistry 34:9599 (1995); and U.S. Pat. Nos.5,773,598; 5,496,938; 5,580,737; 5,654,151; 5,726,017; 5,786,462;5,503,978; 6,028,186; 6,110,900; 6,124,449; 6,127,119; 6,140,490;6,147,204; 6,168,778; and 6,171,795. Aptamers can also be expressed froma transfected vector (Joshi et al., J. Virol. 76:6545 (2002).

Aptamers that bind to a particular target, for example, TRL2 or HMGB,can be selected by using an iterative process called SELEX, which standsfor Systematic Evolution of Ligands by EXponential enrichment (Burke etal., supra; Ellington and Szostak, supra; Schneider et al., supra; Tuerkand Gold, Proc. Natl. Acad. Sci. USA 89:6988 (1992); and Tuerk and Gold,Science 249:505 (1990)). Several variations of SELEX have beendeveloped, which improve the process and allow its use under particularcircumstances. See, e.g., U.S. Pat. Nos. 5,472,841; 5,503,978;5,567,588; 5,582,981; 5,637,459; 5,683,867; 5,705,337; 5,712,375; and6,083,696. Thus, the production of aptamers to a particularoligopeptide, including TLR2 or HMGB, requires no undue experimentation.Screening Methods for Identifying Agents that Inhibit the InteractionBetween HMGB and TLR2

The present invention provides assays for screening candidate inhibitorsor test agents (e.g., a candidate compound) to detect and/or identifythose that inhibit at least one function characteristic of theinteraction between a HMGB and TLR2, as described herein, as well asagents identifiable by the assays. As used herein, a “candidateinhibitor” or “test agent” is a molecule, be it naturally-occurring orartificially-derived, and includes, for example, peptides, proteins,peptidomimetics, synthetic molecules, for example, synthetic organicmolecules, naturally-occurring molecules, for example, naturallyoccurring organic molecules, nucleic acid molecules, and componentsthereof.

In general, test agents for use in the present invention may beidentified from libraries of natural products or synthetic (orsemi-synthetic) extracts or chemical libraries according to methodsknown in the art. Those skilled in the field of drug discovery anddevelopment will understand that the precise source of test extracts orcompounds is not critical to the screening procedure(s) of theinvention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited toplant-, fungal-, prokaryotic- or animal-based extracts, fermentationbroths, and synthetic compounds, as well as modification of existingcompounds. Numerous methods are also available for generating random ordirected synthesis (e.g., semi-synthesis or total synthesis) of anynumber of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic small molecule libraries are commercially available, e.g.,from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical(Milwaukee, Wis.). Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant, and animal extracts are commerciallyavailable from a number of sources, including Biotics (Sussex, UK),Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce,Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).

In addition, natural and synthetically produced molecules or librariescan be generated by any suitable method (e.g., by standard extractionand fractionation methods). For example, candidate compounds can beobtained using any of the numerous approaches in combinatorial librarymethods, including biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to polypeptide libraries, whilethe other four approaches are applicable to polypeptide, non-peptideoligomer or small molecule libraries of compounds (Lam, Anticancer DrugDes., 12:145 (1997)). Furthermore, if desired, any library or compoundcan be readily modified using standard chemical, physical, orbiochemical methods.

It is understood that an inhibitor can inhibit a function characteristicof the interaction between HMGB and TLR2 in varying degrees. Forexample, the inhibitor can decrease the function characteristic of theinteraction between HMGB and TLR2 by at least about 10%, 20%, 40%, 50%,or 75%, or by at least about 90%, relative to an appropriate control.

When a crude extract is found to inhibit a function characteristic ofthe interaction between HMGB and TLR2, further fractionation of thepositive lead extract can be performed to isolate chemical constituentsresponsible for the observed effect. The assays described herein for thedetection and/or identification of activities in mixtures of compoundscan be used to purify the active component or to test derivativesthereof. If desired, compounds shown to be useful agents for treatmentcan be chemically modified according to methods known in the art.Compounds identified as being of therapeutic value may be subsequentlyanalyzed using animal models for diseases in which it is desirable toalter a function characteristic of the interaction between HMGB andTLR2.

Binding Inhibition Assays

Binding inhibition assays can be carried out using any suitable method.Such assays can be used, alone or in combination with each other orother suitable methods, to identify inhibitors of a functioncharacteristic of the interaction between HMGB and TLR2 (e.g., ananti-inflammatory agent). The methods of the present invention can beadapted for high-throughput screening in which large numbers of samplesare processed (e.g., a 96- or 384-well format). Such assays can becarried out using cells (for example, macrophages), cell extracts, orpurified proteins.

Binding inhibition assays can be used to identify agents that inhibitthe interaction between HMGB and TLR2. For example, a binding assay canbe conducted in which the binding of HMGB to TLR2 in the absence of acandidate inhibitor, is compared with the binding of HMGB to TLR2 in thepresence of the candidate inhibitor. TLR2 can be contacted with HMGBligand and the candidate inhibitor simultaneously, or one after theother, in either order. A reduction in the extent of binding of HMGB toTLR2 in the presence of the candidate inhibitor is indicative that thecandidate is an inhibitor.

In one method for detection and/or identification of inhibitors, anagent (e.g., a composition comprising one or more candidate inhibitors)to be tested, TLR2 and HMGB can be maintained under conditions suitablefor binding, and formation of a complex between the HMGB and TLR2 isdetected or measured. For example, the extent of complex formation canbe determined relative to a suitable control (e.g., compared withbackground determined in the absence of the candidate inhibitor, orcompared with complex formation with a second agent (i.e., a standard)).

An interaction (e.g., complex formation) between HMGB and TLR2 can bedetected directly (for example, by measuring the binding affinity, usingstandard methods. In one embodiment, the TLR2, candidate inhibitor, orthe HMGB can be labeled with a suitable label (e.g., fluorescent moiety,chemiluminescent group, epitope tag, radioisotope, enzyme label, oraffinity tag), and complex formation can be determined indirectly bydetection of the label. Specificity of binding can be assessed bycompetition or displacement, for example, using a known TLR2 or HMGBligand (e.g., those described herein) as a competitor.

In another example, a fusion protein comprising HMGB and a detectablelabel, for example, a fluorescent protein, such as green fluorescentprotein, or a hapten that can be detected with an antibody (e.g.,labeled antibody), such as glutathione S-transferase or biotin iscontacted with TLR2. The TLR2 can be immobilized on a solid support, forexample, a microtiter well. Preferably, the TLR2 and fusion protein arein a solution suitable for complex formation between TLR2 and HMGB, asdescribed herein. The fusion protein binds to TLR2. Unbound fusionprotein can be removed, for example, by washing the solid support or byother means suitable for separation of unbound fusion protein from boundfusion protein complexed with TLR2. The complex formed between thefusion protein and TLR2 is then indirectly detected, for example, bymeasuring fluorescence of bound fluorescent protein, or by contactingthe complex with an antibody that binds the hapten portion of the fusionprotein, using, for example, an ELISA assay or a radioimmunoassay. Theassay can be repeated in the presence of a candidate inhibitor. If thecandidate inhibitor disrupts complex formation between the TLR2 bindingfusion protein and TLR2, then a decrease in the signal can be detecteddue to decreased complex formation between the fusion protein and TLR2.Such a decrease indicates that the candidate inhibitor is an inhibitorof the ineraction between HMGB and TLR2. Alternatively, the HMGB fusionpolypeptide can be immobilized on a solid support, and the TLR2 andcandidate inhibitor is added and assessed for complex formation.

In another example, a fusion protein comprising HMGB and an enzyme, forexample, β-galactosidase, luciferase, chloramphenicol acetyltransferase, or alkaline phosphatase, is contacted with TLR2. The TLR2can be immobilized on a solid support, for example, a microtiter well.Preferably, the TLR2 and fusion protein are in a solution suitable forcomplex formation between TLR2 and HMGB, as described herein. The fusionprotein will bind to TLR2. Unbound fusion protein can be removed, forexample, by washing the solid support or by other means suitable forseparation of unbound fusion protein from bound fusion protein complexedwith TLR2. The complex formed between the fusion protein and TLR2 can beindirectly detected, for example, by adding the appropriate substratefor the enzyme present in the fusion protein, and detecting enzymeactivity using, for example, using a microplate reader or a luminometer.The assay can be repeated in the presence of a candidate inhibitor. Ifthe candidate inhibitor disrupts complex formation between the fusionprotein and TLR2, then a decrease in enzyme activity due to decreasedcomplex formation between the fusion protein and TLR2 can be detected.Such a decrease indicates that the candidate inhibitor is an inhibitorof TLR2 binding.

Another method for identifying an inhibitor of the interaction betweenHMGB and TLR2 is through phage display techniques. For example, HMGB canbe expressed on the surface of phage (e.g., as a fusion protein with aphage coat protein). The phage is then contacted with TLR2 (immobilized,for example, on a solid support, such as a microtiter well) and thephage binds to the TLR2. The complex can be detected using any suitablemethod. For example, the complex can be contacted with an antibody thatrecognizes the phage, and binding of the antibody can be detected using,for example, an ELISA assay or a radioimmunoassay. The assay can berepeated in the presence of a candidate inhibitor. If the candidateinhibitor disrupts complex formation between the HMGB displayed on thephage, then a decrease in signal (e.g., the amount of antibody binding)can be detected due to decreased complex formation between the fusionand TLR2. Such a decrease indicates that the candidate inhibitor is aninhibitor of TLR2 binding.

Cell based assays can also be used to detect and/or identify inhibitorsof the interaction between HMGB and TLR2. For example, a yeasttwo-hybrid system such as that described by Fields and Song (Fields andSong, Nature 340:245-246 (1989)) can be used to identify polypeptidesthat interact with TLR2. In such a yeast two-hybrid system, vectors areconstructed based on the flexibility of a transcription factor that hastwo functional domains (a DNA binding domain and a transcriptionactivation domain). If the two domains are separated but fused to twodifferent proteins that interact with one another, transcriptionalactivation can be achieved, and transcription of specific markers (e.g.,nutritional markers such as His and Ade, or color markers such as lacZ)can be used to identify the presence of interaction and transcriptionalactivation. For example, in the methods of the invention, a first vectoris used that includes a nucleic acid encoding a DNA binding domain and aTL2 receptor polypeptide or an HMGB polypeptide, or functionalequivalent or derivative thereof, and a second vector is used thatincludes a nucleic acid encoding a transcription activation domain and anucleic acid encoding a polypeptide that potentially may interact withthe TLR2 polypeptide or HMGB polypeptide of interest, or functionalequivalent or derivative thereof. Incubation of yeast containing thefirst vector and the second vector under appropriate conditions (e.g.,mating conditions such as used in the MATCHMAKER™ system from Clontech)allows identification of colonies that express the markers of thepolypeptide(s). These colonies can be examined to identify thepolypeptide(s) that interact with the TLR2 polypeptide or HMGBpolypeptide encoded by the nucleic acid molecule or a functionalequivalent or derivative thereof. Such polypeptides may be useful ascompounds that bind to HMGB or TLR2 and inhibit the interaction betweenTLR2 and HMGB and can be further tested using, for example, assaysdescribed herein.

Inhibitors of the interaction between HMGB and TLR2 can also beidentified by measuring release of a proinflammatory cytokine from acell, for example, a macrophage, or MyD 88 activity or NF-κB activity inan ex vivo assay. For example, an interaction between HMGB and TLR2results in the release of proinflammatory cytokines from the cell, whichcan be detected and measured using, for example, ELISA kits. MyD 88 orNF-κB activity can be assessed as described herein, using commerciallyavailable kits, or using other methods known to one skilled in the art.The assay can be repeated in the presence of a candidate inhibitor. Ifthe candidate inhibitor disrupts the interaction between the HMGB andTLR2, then a decrease in cytokine release, MyD 88, activity, or NF-κBactivity can be detected due to decreased interaction between HMGB andTLR2. Such a decrease indicates that the candidate inhibitor is aninhibitor of the interaction of HMGB with TLR2.

In certain embodiments it may be desirable to immobilize (directly orindirectly) a component of the assay on a matrix or other solid support,in order to facilitate separation of bound from unbound components ofthe assay, as well as to accommodate automation of the assay. Theabove-described assays can be carried out in any suitable manner forcombining the reactants. For example, the components may be combined ina suitable vessel such as a microtiter plate, test tube, ormicro-centrifuge tube. In one embodiment, a fusion protein (e.g., aglutathione-S-transferase fusion protein) can be provided that adds adomain that allows a component of the assay to be bound to a matrix orother solid support.

Detection and/or identification of an inhibitor of complex formationbetween a HMGB and TLR2 can also occur through the use of “in silico”screening methods, which involve the use of computer programs to testthe docking of candidate inhibitors. Such methods comprise determiningfunctional residues of HMGB involved in forming a complex with TLR2;developing one or more three-dimensional structures based on thefunctional residues identified in the previous step using any suitablemethod; comparing the one or more three-dimensional structures with oneor more test agents having calculatable tertiary structures; andidentifying agents having a spatial orientation consistent with forminga complex with TLR2 and inhibiting complex formation between TLR2 andHMGB. Agents identified in this manner can be further assessed foractivity using, for example, assays described herein, and can be used asanti-inflammatory agents.

Information regarding the HMGB functional residues involved in forming acomplex with TLR2 can be used to develop one or more three-dimensionalstructures with which a successful test agent (i.e., an inhibitor of theinteraction between HMGB and TLR2) comes into contact with TLR2. Thethree-dimensional structures are compared with or tested against one ormore test agents that have calculatable tertiary structures. Forexample, computer programs in which one or more test agents areindividually docked to TLR2 and examined for suitable spatialorientation with respect to the TLR2 can be used to test for anappropriate test agent. A test agent has suitable spatial orientation ifthe test agent binds to TLR2 with favorable energy, as determined usingthe parameters of the computer software used to detect docking of testagents and associated binding energies. Successful compounds, orderivatives thereof can then be tested using in vitro or in vivo assays,for example, as described herein.

This invention further pertains to novel compounds identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, a compound identified asdescribed herein (e.g., a candidate compound that is an inhibitor ofcomplex formation between a HMGB and TLR2) can be used in an animalmodel to determine the efficacy, toxicity, or side effects of treatmentwith such a compound, for example, as described below.

Pharmaceutical Compositions

The present invention is also directed to a composition comprising anagent that inhibits the interaction of HMGB and TLR2 in apharmaceutically acceptable carrier. Preferably, the agent binds to TLR2and inhibits binding by HMGB. Thus, the composition and methodsdisclosed herein can be used to inhibit an inflammatory condition. Thecondition can be one where the inflammatory cytokine cascade causes asystemic reaction, such as with endotoxic shock. Alternatively, thecondition can be mediated by a localized inflammatory cytokine cascade,as in rheumatoid arthritis. Preferably, the inflammatory condition isappendicitis, peptic, gastric or duodenal ulcers, peritonitis,pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis,diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis,hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma,allergy, anaphylactic shock, immune complex disease, organ ischemia,reperfusion ischemia, organ necrosis, hay fever, sepsis, septicemia,endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma,granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis,prostatitis, urethritis, bronchitis, emphysema, rhinitis, cysticfibrosis, pneumonitis, pneumoultramicroscopicsilicovolcanoconiosis,alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza,respiratory syncytial virus infection, herpes infection, HIV infection,hepatitis B virus infection, hepatitis C virus infection, disseminatedbacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis,hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria,warts, wheals, vasulitis, angiitis, endocarditis, arteritis,atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardialischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease,coeliac disease, congestive heart failure, adult respiratory distresssyndrome, meningitis, encephalitis, multiple sclerosis, cerebralinfarction, cerebral embolism, Guillame-Barre syndrome, neuritis,neuralgia, spinal cord injury, paralysis, uveitis, arthritides,arthralgias, osteomyelitis, fasciitis, Paget's disease, gout,periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis,thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome,Behcets's syndrome, chronic obstructive pulmonary disease, psoriasis,allograft rejection, graft-versus-host disease, Type I diabetes,ankylosing spondylitis, Berger's disease, Retier's syndrome, or Hodgkinsdisease. In one embodiment, the condition is not sepsis. In anotherembodiment, the condition is not a bacterial infection or bacterialsepsis. In another embodiment, the condition is not septic shock,rosecea, acne, shock, a viral infection, toxic shock, acuteinflammation, chronic inflammation, atopic dermatitis, chronicobstructive pulmonary disease, or intestinal inflammation.

In more preferred embodiments, the condition is peritonitis,pancreatitis, ulcerative colitis, Crohn's disease, organ ischemia,reperfusion ischemia, cachexia, burns, myocardial ischemia, adultrespiratory distress syndrome, multiple sclerosis, restenosis,rheumatoid arthritis, systemic lupus erythematous, Behcet's syndrome,psoriasis, allograft rejection and graft-versus-host disease. Where thecondition is allograft rejection, the composition may advantageouslyalso include an immunosuppressant that is used to inhibit allograftrejection, such as cyclosporin.

The carrier included with the polypeptide in these compositions ischosen based on the expected route of administration of the compositionin therapeutic applications. The route of administration of thecomposition depends on the condition to be treated. For example,intravenous injection may be preferred for treatment of a systemicdisorder such as endotoxic shock, and oral administration may bepreferred to treat a gastrointestinal disorder such as a gastric ulcer.The route of administration and the dosage of the composition to beadministered can be determined by the skilled artisan without undueexperimentation in conjunction with standard dose-response studies.Relevant circumstances to be considered in making those determinationsinclude the condition or conditions to be treated, the choice ofcomposition to be administered, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms. Thus,depending on the condition, the antibody composition can be administeredorally, parenterally, intranasally, vaginally, rectally, lingually,sublingually, bucally, intrabuccaly, and transdermally to the patient.

Accordingly, compositions designed for oral, lingual, sublingual,buccal, and intrabuccal administration can be made without undueexperimentation by means well known in the art, for example, with aninert diluent or with an edible carrier. The compositions may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the pharmaceutical compositions ofthe present invention may be incorporated with carriers and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth, or gelatin. Examples of carriers includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin, and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring, and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and non-toxic in the amounts used.

Methods of Therapy

Inhibition of the binding of HMGB to TLR2 according to the presentinvention, provides an effective way of inhibiting TLR2-binding-mediatedfunctions, for example, inflammation and/or release of a proinflammatorycytokine from a cell. Thus, agents that inhibit the interaction betweenTLR2 and HMGB, including inhibitors such as those identified asdescribed herein, can be used as agents to treat inflammatoryconditions, for therapeutic purposes.

The route of administration and the dosage of the agent orpharmaceutical composition to be administered can be determined by theskilled artisan without undue experimentation in conjunction withstandard dose-response studies. Relevant circumstances to be consideredin making those determinations include the condition or conditions to betreated, the choice of composition to be administered, the age, weight,and response of the individual patient, and the severity of thepatient's symptoms. Typically, an effective amount can range from 0.01mg per day to about 100 mg per day for an adult. Preferably, the dosageranges from about 1 mg per day to about 100 mg per day or from about 1mg per day to about 10 mg per day.

In one aspect, the present invention provides a method of treating aninflammatory condition in an individual, or treating an individual atrisk for having an inflammatory condition, comprising administering tothe individual an effective amount of an agent that inhibits theinteraction between HMGB and TLR2 to an individual in need of suchtherapy. In one embodiment, the agent binds TLR2 and inhibits binding byHMGB. As used herein, an “effective amount” is an amount sufficient toprevent or decrease an inflammatory response, or to improve aninflammatory condition. Methods for assessing inflammatory responses andconditions are known in the art.

The terms “therapeutic” and “treatment” as used herein, refer toameliorating symptoms associated with a disease or condition, includingpreventing or delaying the onset of the disease symptoms, and/orlessening the severity or frequency of symptoms of the disease orcondition. The term “individual” is defined herein to include animalssuch as mammals, including, but not limited to, primates, cows, sheep,goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or otherbovine, ovine, equine, canine, feline, rodent, or murine species. In oneembodiment, the animal is a human. Diseases and conditions associatedwith inflammation can be treated using the method.

Inflammatory diseases or conditions that can be treated with inhibitorsof the binding of HMGB to TLR2 are described herein.

Modes of Administration

The pharmaceutical compositions of the present invention can beadministered parenterally such as, for example, by intravenous,intramuscular, intrathecal, or subcutaneous injection. Parenteraladministration can be accomplished by incorporating the antibodycompositions of the present invention into a solution or suspension.Such solutions or suspensions may also include sterile diluents such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol, or other synthetic solvents. Parenteralformulations may also include antibacterial agents such as, for example,benzyl alcohol or methyl parabens, antioxidants such as, for example,ascorbic acid or sodium bisulfite and chelating agents such as EDTA.Buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose may also beadded. The parenteral preparation can be enclosed in ampules, disposablesyringes, or multiple dose vials made of glass or plastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the antibody composition in the glycerin, mixing the heatedglycerin after which purified water may be added, and pouring the hotmixture into a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches,ointments, creams, gels, salves, and the like.

The present invention includes nasally administering to the mammal atherapeutically effective amount of the pharmaceutical composition. Asused herein, nasally administering or nasal administration includesadministering the composition to the mucous membranes of the nasalpassage or nasal cavity of the patient. As used herein, pharmaceuticalcompositions for nasal administration of a composition includetherapeutically effective amounts of the agonist prepared by well-knownmethods to be administered, for example, as a nasal spray, nasal drop,suspension, gel, ointment, cream, or powder. Administration of thecomposition may also take place using a nasal tampon or nasal sponge.

The therapeutic compositions described herein can also include anantagonist of an early sepsis mediator. As used herein, an early sepsismediator is a proinflammatory cytokine that is released from cells soon(i.e., within 30-60 min.) after induction of an inflammatory cytokinecascade (e.g., exposure to LPS). Nonlimiting examples of these cytokinesare TNF, IL-1α, IL-1β, IL-6, PAF, and MIF. Also included as early sepsismediators are receptors for these cytokines (for example, tumor necrosisfactor receptor type 1) and enzymes required for production of thesecytokines, for example, interleukin-1β converting enzyme). Antagonistsof any early sepsis mediator, now known or later discovered, can beuseful for these embodiments by further inhibiting an inflammatorycytokine cascade.

EXEMPLIFICATION

To examine the effects of HMGB1 or HMGB1 B Box on the NF-κB-dependentELAM promoter, the following experiment was carried out. RAW 264.7macrophages were transiently co-transfected with an expression plasmidencoding a murine MyD 88-dominant-negative (DN) mutant (corresponding toamino acids 146-296), or empty vector, plus a luciferase reporterplasmid under the control of the NF-κB-dependent ELAM promoter, asdescribed by Means et al. (J. Immunol. 166:4074-4082 (2001)). A portionof the cells were then stimulated with full-length HMBG1 (100 ng/ml), orpurified HMGB1 B box (10 μg/ml), for 5 hours. Cells were then harvestedand luciferase activity was measured, using standard methods. Alltransfections were performed in triplicate, repeated at least threetimes, and a single representative experiment is shown in FIG. 1A. Asshown in FIG. 1A, HMGB1 stimulated luciferase activity in samples thatwere not co-transfected with the MyD 88 dominant negative, and the levelof stimulation was decreased in samples that were co-transfected withthe MyD 88 dominant negative. This effect was also observed in samplesadministered HMGB B box.

The effect of HMGB1 or HMGB1 B box on NF-κB activation was alsoexamined. CHO reporter cell lines that constitutively express humanToll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) have beenpreviously described (Means et al., J. Immunology, 163:3920-3927(1999)). These reporter lines also contain a stably transfectedELAM-CD25 reporter gene, and express human CD25 on their surface as aconsequence of NF-κB activation. CHO/TLR2 and CHO/TLR4 cells werestimulated with IL-1 (10 ng/ml), purified full-length HMG-1 (100 ng/ml),or purified B box (10 μg/ml) for 18 hours. Following stimulation, cellswere stained with a PE-labeled anti-CD25 monoclonal antibody and surfaceexpression of CD25 was measured by flow cytometry. The results of thisstudy are shown in FIG. 1B. Data are expressed as the ratio(fold-activation) of the percent of CD25⁺ cells in unstimulated andstimulated cell populations that were gated to exclude the lowest 5% ofcells based on mean FL1 fluorescence. In CHO/TLR2 cells, stimulationwith each of HMGB1 and HMGB1 B box resulted in increased CD25 expressioncompared to the CHO/TLR4 samples.

The effect of anti-RAGE antibodies, anti-TLR2 antibodies, a combinationof anti-RAGE antibodies and anti-TLR2 antibodies or IgG, onHMG-1-mediated TNF release in RAW 264.7 cells was also determined. RAW264.7 cells were seeded into 24-well tissue culture plates and used at90% confluence. Cells were incubated with HMG-1 with or withoutanti-RAGE antibody (Cat# sc-8230, Santa Cruz Biotech Inc., Santa Cruz,Calif.), anti-TLR2 antibody (Affinity-purified polyclonal antibody, Cat# sc-12504, D17, Santa Cruz) or IgG (non-immune IgG, Sigma, St. Louis,Mo.) in Optimum I medium (Life Technologies, Grand Island, N.Y.) in thepresence of polymyxin B (100 units/ml, Sigma, St. Louis, Mo.) for 16hours. Antibodies were dialyzed against PBS to remove sodium azidebefore use. Conditioned media were collected and a TNF ELISA wasperformed, using standard ELISA methods. Data (n=3) were expressed as apercentage of the activity achieved with HMG-1 alone. The results ofthis study are shown in FIG. 1C. Both anti-RAGE and anti-TLR2 antibodiessignificantly (*P<0.05) inhibited HMG-1-mediated TNF release.Combination of the 2 antibodies had additive effects in inhibiting TNFrelease whereas IgG was irrelevant.

Toll-like receptor (TLR) proteins are highly conserved receptors thatactivate innate immune cells in response to a variety of endogenous andexogenous stimuli (Aderem and Ulevitch, Nature, 406:782-787 (2000)). Toinvestigate whether HMGB1 signals through Toll-like receptors to releasecytokine, macrophage-like RAW 264.7 cells stimulated with HMGB1 in thepresence of anti-TLR2 antibody were used. Anti-TLR2 antibody causedapproximately a 50% reduction in HMGB1-induced TNF release (Table 1),indicating that TLR2 participates in the inflammatory response of HMGB1.TABLE 1 TNF release (% compared to TNF release by Stimulus HMGB1 alone)HMGB1 (0.1 μg/ml) 100% HMGB1 (0.1 μg/ml) + anti-TLR2 (2.5 μg/ml) 53 ±13% HMGB1 (0.1 μg/ml) + anti-RAGE (2.5 μg/ml) 58 ± 10% HMGB1 (0.1μg/ml) + anti-TLR2 + anti-RAGE 48 ± 10% (2.5 μg/ml each) HMGB1 (0.1μg/ml) + IgG ((2.5 μg/ml) 95 ± 6% 

To further examine the involvement of TLR2 in HMGB1 signaling, culturesof human embryonic kidney HEK cells stably transfected with fusionproteins consisting of CFP (cyan fluorescent protein) fused to eitherTLR2 or TLR4 at the C-terminus were used (provided by Drs. DouglasGolenbock and Eicke Latz, University of Massachusetts, School ofMedicine, Boston, Mass.). The cell lines were cultured in in DMEM mediumsupplemented with 10% FBS, 1× penicillin/streptomycin, 1% L-glutamine,500 μg/ml geneticin, and were used in experiments at 90% confluence.Experiments using these cell lines were performed under serum-freeconditions.

HMGB1 was produced in E. coli, as described by Wang et al. (Science,285:248-251 (1999)). Briefly, a recombinant plasmid encoding rat HMGB1was transformed into the protease-deficient E. coli strain BL21(Novagen, Madison, Wis.) and grown in LB medium containing ampicillin(50 μg/ml), and fusion protein expression was induced by addition of 1mM IPTG for 3 hours at 37° C. Bacteria were sonicated in ice-cold PBSplus 1× protease inhibitor cocktail and 1 mM PMSF. HMGB1 was purifiedwith a calmodulin-binding resin column (Stratagene, La Jolla, Calif.)and passed over a polymyxin B column (Pierce, Rockford, Ill.) to removepossibly contaminating LPS. The integrity of protein was verified bySDS-PAGE with Coomassie Blue staining, with the purity predominantlyover 85%. The HMGB1 protein was administered to the HEK-pcDNA (vectorcontrol), HEK-TLR2, and HEK-TLR4 cell lines at various concentrations,as indicated in FIG. 2A and release of IL-8 from the cells was measuredusing a commercially available ELISA kit from R&D Systems Inc.(Minneapolis, Minn.) according to the manufacturer's instructions. Asshown in FIG. 2A, HMGB1 dose dependently stimulated IL-8 release incells over-expressing TLR2 but not in cells over-expressing TLR4.

To ascertain that the effects of HMGB1 are not due to the possibleminute amount of contaminants from bacteria, HMGB1 was expressed inmammalian CHO cells, to produce HMGB1 that was free of bacterialcomponents. The CHO cell line was produced as follows. An N-terminal 3×Flag-tagged rat HMGB1 nucleic acid fragment was cloned into the plasmidpIRES2-EGFP (Clontech, Palo Alto, Calif.) to generate plasmid psF-HMGB1using standard molecular biology techniques. A eukaryotic expressionplasmid (psF-HMGB1) was engineered to secrete an N-terminal, 3×Flag-tagged rat HMGB1 recombinant protein. Plasmid psF-HMGB1 wastransfected into CHO cells using the calcium phosphate method(Gibco-BRL, Grand Island, N.Y.) according to the manufacturer'sinstructions. Adherent CHO cells were grown in α-MEM media supplementedwith 10% FBS and 2 mM glutamine. Transfected cells were selected for 10days in 600 μg/ml active Geneticin. Stably transfected cells were FACSsorted for GFP (green fluorescent protein) expression and GFP-expressingcell lines were cloned by limiting dilution. A high HMGB1 secreting cellline (CHO HMGB1) was adapted to growth in suspension by using CHO-S-SFMII media supplemented with 300 μg/ml Geneticin and 1×penicillin/streptomycin. Fusion HMGB1 expression was about 0.5 μg/mlmedium. HMGB1 protein was isolated from conditioned supernatant byaffinity purification using flag antibody (ANTI-FLAG 2 affinity gel)according to the manufacturer's instructions (Sigma, St. Louis, Mo.).

The HMGB1 protein was administered to the HEK-TLR2 cell line at variousdoses, as indicated in FIG. 2B and release of IL-8 from the cells wasmeasured using a commercially available ELISA kit from R&D Systems Inc.(Minneapolis, Minn.). As shown in FIG. 2B, recombinantly produced CHOcell derived HMGB1 stimulated IL-8 release from HEK-TLR2 cells. Theseresults indicate that HMGB1 is an endogenous agonist of TLR2.

To ascertain the specificity of HMGB1 signaling, HEK-TLR2 cells weretreated with nothing, HMGB1 alone, HMGB1 digested by trypsin, HMGB1 plusanti-HMGB1 antibody, HMGB1 plus IgG, or LPS alone, as indicated in FIG.3A, and release of IL-8 from the cells was measured using a commerciallyavailable ELISA kit from R&D Systems Inc. (Minneapolis, Minn.). HMGB1was trypsin-treated as follows. Trypsin-EDTA was added to purified HMGB1(500 μg/ml in PBS) at 0.05% (final concentration), and digestion wascarried out at 25° C. overnight. Proteins were visualized by Coomassieblue staining, and degradation of proteins was verified by SDS-PAGEbefore and after digestion. As shown in FIG. 3A, trypsin treatment ofHMGB1 abolished HMGB1-induced IL-8 release, indicating that the effectof HMGB1 was specific. The anti-HMGB1 antibody significantly inhibitedIL-8 release in cell cultures exposed to HMGB1. This data providesfurther evidence of the specificity of HMGB signaling. LPS, a known TLR4agonist, stimulated IL-8 release in HEK-TLR4 cells administered LPS(FIG. 3B) but not in HEK-TLR2 cells, verifying that the transfected TLRproteins function similarly to endogenous TLR proteins in signaltransduction. These results also show that residual amounts of LPS inHMGB preparations do not account for the observed effects ofadministration of HMGB to HEK-TLR2 cells.

TLR proteins transduce signals through an intermediate protein, MyD88(Ombrellino et al., Lancer, 354:1446-1447 (2000)). To examine thepossible role of MyD88 in mediating HMGB1 signaling, RAW 264.7macrophages were co-transfected with plasmids encoding a dominantnegative mutant MyD88 protein and an NF-κB-dependent luciferasereporter. The cells were then stimulated with HMGB1. The dominantnegative MyD88 mutant significantly inhibited activation of NF-κBluciferase activity induced by HMGB1 (FIG. 4A). To address thespecificity of the MyD88-dependent response, HMGB1 was added to culturesof CHO cells engineered to constitutively express either human TLR2 orTLR4 (FIG. 4B). These cells also contained an NF-κB-dependent CD25reporter gene as previously described (Yoshimura et al., J. Immunol.,163: 1-5 (1999), and Delude et al., Journal of Immunology, 161:3001-3009(1998)). HMGB1 failed to increase CD25 expression in TLR4-expressingcells, but significantly stimulated CD25 expression in CHO cellsexpressing TLR2, indicating that stimulation through TLR2 by HMGB1 isspecific.

To further study whether HMGB1 signals via TLR2, macrophage-like RAW264.7 cells were labeled with fluorescein isothiocyanate (FITC)-taggedHMGB1, produced using a commercially available kit (Pierce, Rockford,Ill.) according to the manufacturer's instructions. The labeling wascarried out as follows. The cells were plated in 8-well slide chambers(Lab-Tek, Nalge Nunc Internationals, Naperville, Ill.) and used at 70%confluence. The cells were either left alone, or were pre-incubated inthe presence of anti-TLR2 antibody or anti-TLR4 antibody (1 μg/ml) for20 minutes at 37° C. in Opti-MEM I medium. FITC-labeled HMGB1 (1 μg/ml)was then added for an additional 15 minutes at 37° C. Cells were washed3 times with PBS and fixed for 15 minutes at room temperature in 4%paraformaldehyde-PBS solution (pH 7.2). After fixing, cells were washedwith PBS once and mounted for viewing by fluorescent confocalmicroscopy. Uptake of FITC-HMGB1 was observed in macrophages incubatedwith FITC-HMGB1 for 15 minutes at 37° C. (FIG. 5A). Pre-treatment withanti-TLR2 antibody markedly reduced the uptake as revealed by thedecrease in cell-associated fluorescence (FIG. 5B). In contrast,pre-treatment with anti-TLR4 antibody did not have any effects onFITC-HMGB1 uptake (FIG. 5C) indicating that the effect of anti-TLR2 onblocking HMGB1 uptake was specific and thus further support that HMGB1signals via TLR2.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating an inflammatory condition in an individual, comprising administering to said individual an effective amount of an agent that inhibits the interaction between a high mobility group B (HMGB) polypeptide or functional equivalent thereof and Toll-like receptor 2, with the proviso that said agent is not an antibody that binds to an HMGB1 polypeptide.
 2. The method of claim 1, wherein said agent binds said Toll-like receptor 2 and inhibits binding by a high mobility group B (HMGB) polypeptide or functional equivalent thereof.
 3. The method of claim 1, wherein said HMGB polypeptide is mammalian.
 4. The method of claim 3, wherein said HMGB polypeptide is HMGB1.
 5. The method of claim 1, wherein said inflammatory condition is selected from the group consisting of peritonitis, pancreatitis, ulcerative colitis, Crohn's disease, asthma, organ ischemia, reperfusion ischemia, sepsis, cachexia, burns, myocardial ischemia, adult respiratory distress syndrome, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematous, chronic obstructive pulmonary disease, psoriasis, Behcet's syndrome, allograft rejection and graft-versus-host disease.
 6. The method of claim 1, wherein said agent is soluble Toll-like receptor
 2. 7. A method for inhibiting the release of a proinflammatory cytokine from a cell, comprising administering to said cell an effective amount of an agent that inhibits the interaction between an HMGB polypeptide or functional equivalent thereof and a Toll-like receptor 2, with the proviso that said agent is not an antibody that binds to an HMGB1 polypeptide.
 8. The method of claim 7, wherein said agent binds a Toll-like receptor 2 and inhibits binding by an HMGB polypeptide.
 9. The method of claim 7, wherein said HMGB polypeptide is mammalian.
 10. The method of claim 9, wherein said HMGB polypeptide is HMGB1.
 11. The method of claim 7, wherein said cell is a macrophage.
 12. The method of claim 7, wherein said proinflammatory cytokine is selected from the group consisting of tumor necrosis factor (TNF), interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6).
 13. The method of claim 7, wherein said cell is in an individual having or at risk for an inflammatory condition.
 14. The method of claim 13, wherein said inflammatory condition is selected from the group consisting of peritonitis, pancreatitis, ulcerative colitis, Crohn's disease, asthma, organ ischemia, reperfusion injury, sepsis, cachexia, burns, myocardial ischemia, adult respiratory distress syndrome, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematous, Behcet's syndrome, allograft rejection and graft-versus-host disease.
 15. The method of claim 7, wherein said agent is soluble Toll-like receptor
 2. 16. A method of determining whether an agent inhibits inflammation, comprising: (a) contacting a Toll-like receptor 2 with said agent and an HMGB polypeptide or a functional equivalent thereof; and (b) detecting binding of an HMGB polypeptide to said Toll-like receptor 2, wherein an agent that decreases binding of said HMGB polypeptide to said Toll-like receptor 2 relative to a suitable control is an agent that inhibits inflammation.
 17. The method of claim 16, wherein said HMGB polypeptide or functional equivalent thereof is mammalian.
 18. The method of claim 17, wherein said HMGB polypeptide is HMGB1.
 19. The method of claim 16, wherein said Toll-like receptor 2 is on the surface of a cell.
 20. The method of claim 19, wherein said cell is a macrophage. 